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Objective: To assess the clinical performance of the human papillomavirus (HPV) 16 18/45 genotype assay (AHPV-GT) combined with age stratification in triaging women with histologic low-grade squamous intraepithelial lesion/cervical intraepithelial neoplasia grade 1 (LSIL [CIN1]) preceded by HPV E6/E7 mRNA assay (Aptima HPV, AHPV) positive/normal or mildly abnormal cytology. Design: Longitudinal study. Setting: Gynaecological clinic in Huzhou Maternity & Child Health Care Hospital, China. Population: Women aged ≥21 years with histologic LSIL (CIN1) preceded by AHPV-positive/normal or mildly abnormal cytology. Methods: Women underwent AHPV-GT testing at baseline and were followed at 6-month intervals for up to 2 years. At each follow-up, women with abnormal cytology or AHPV positivity were referred for colposcopy and then biopsy if indicated. Main outcome measures: The 2-year cumulative incidence rates (CIRs) of CIN3+ stratified by baseline AHPV-GT genotyping. Results: Of 349 eligible women, 25 women (7.2%) progressed, 301 (86.2%) regressed, and 23 (6.6%) persisted during the follow-up. The 2-year CIRs of CIN3+ in AHPV-GT-positive women were both significantly higher than those in AHPV-GT-negative women overall (8.6% vs. 1.7%, P = 0.014) and in the ≥25-year-old group (10.9% vs. 1.5%, P = 0.002) but slightly higher in the 21–24-year-old group ( P > 0.05). Conclusions: AHPV-GT testing with age stratification is effective for triaging women with histologic LSIL (CIN1) preceded by AHPV-positive/normal or mildly abnormal cytology. Immediate treatment is a rational recommendation for women ≥ 25 years old with histologic LSIL (CIN1) preceded by AHPV-GT positivity when good surveillance is not assured.

Butenyl-spinosyn is a highly effective and broad-spectrum biopesticide produced by Saccharopolyspora pogona. However, the yield of this compound is difficult to increase because the regulatory mechanism of secondary metabolism is still unknown. Here, the transcriptional regulator Sp13016 was discovered to be highly associated with butenyl-spinosyn synthesis and bacterial growth. Overexpression of sp13016 improved butenyl-spinosyn production to a level that was 2.84-fold that of the original strain, while deletion of sp13016 resulted in a significant decrease in yield and growth inhibition. Comparative proteomics revealed that these phenotypic changes were attributed to the influence of Sp13016 on the central carbon metabolism pathway to regulate the supply of precursors. Our research helps to reveal the regulatory mechanism of butenyl-spinosyn biosynthesis and provides a reference for increasing the yield of natural products of Actinomycetes.

Reduction and optimization of the microbial genome is an important strategy for constructing synthetic biological chassis cells and overcoming obstacles in natural product discovery and production. However, it is of great challenge to discover target genes that can be deleted and optimized due to the complicated genome of actinomycetes. Saccharopolyspora pogona can produce butenyl-spinosyn during aerobic fermentation, and its genome contains 32 different gene clusters. This suggests that there is a large amount of potential competitive metabolism in S. pogona, which affects the biosynthesis of butenyl-spinosyn. By analyzing the genome of S. pogona, six polyketide gene clusters were identified. From those, the complete deletion of clu13, a flaviolin-like gene cluster, generated a high butenyl-spinosyn-producing strain. Production of this strain was 4.06-fold higher than that of the wildtype strain. Transcriptome profiling revealed that butenyl-spinosyn biosynthesis was not primarily induced by the polyketide synthase RppA-like but was related to hypothetical protein Sp1764. However, the repression of sp1764 was not enough to explain the enormous enhancement of butenyl-spinosyn yields in S. pogona-Δclu13. After the comparative proteomic analysis of S. pogona-Δclu13 and S. pogona, two proteins, biotin carboxyl carrier protein (BccA) and response regulator (Reg), were investigated, whose overexpression led to great advantages of butenyl-spinosyn biosynthesis. In this way, we successfully discovered three key genes that obviously optimize the biosynthesis of butenyl-spinosyn. Gene cluster simplification performed in conjunction with multiomics analysis is of great practical significance for screening dominant chassis strains and optimizing secondary metabolism. This work provided an idea about screening key factors and efficient construction of production strains.

Lithium-sulfur batteries are promising next-generation energy storage systems with high theoretical specific capacity. Despite extensive research efforts, it is still challenging to rationally design electrocatalysts with fast kinetics and effective adsorption of polysulfides. Herein, Fe-doped ReS

Spinosyns, the secondary metabolites produced by Saccharopolyspora spinosa, are the active ingredients in a family of novel biological insecticides. Although the complete genome sequence of S. spinosa has been published, the transcriptome of S. spinosa remains poorly characterized. In this study, high-throughput RNA sequencing (RNA-seq) technology was applied to dissect the transcriptome of S. spinosa. Through transcriptomic analysis of different periods of S. spinosa growth, we found large numbers of differentially expressed genes and classified them according to their different functions. Based on the RNA-seq data, the CRISPR-Cas9 method was used to knock out the PEP phosphonomutase gene (orf 06952-4171). The yield of spinosyns A and D in S. spinosa-ΔPEP was 178.91 mg/L and 42.72 mg/L, which was 2.14-fold and 1.76-fold higher than that in the wild type (83.51 and 24.34 mg/L), respectively. The analysis of the mutant strains also verified the validity of the transcriptome data. The deletion of the PEP phosphonomutase gene leads to an increase in pyruvate content and affects the biosynthesis of spinosad. The replenishment of phosphoenol pyruvate in S. spinosa provides the substrate for the production of spinosad. We envision that these transcriptomic analysis results will contribute to the further study of secondary metabolites in actinomycetes.

Background Etiological diagnosis is a key step in the treatment of patients with rare pulmonary mycosis, and the lack of understanding of this disease and lack of specific markers for the detection of rare species, such as Exophiala dermatitidis, add to the difficulty in diagnosing the condition. Therefore, improving the diagnostic strategies for this disease is very important. Case presentation A 52-year-old man presented with cough, sputum production and hemoptysis; chest computed tomography (CT) revealed multiple bilateral lesions. The pathogen was unable to be identified after three biopsies. Subsequently, we performed combined tissue metagenomic next-generation sequencing (mNGS). The results of mNGS and a good therapeutic response helped to identify the causative pathogen as Exophiala dermatitidis. Finally, the patient was diagnosed with Exophiala dermatitidis pneumonia. Conclusions Combining molecular techniques, such as mNGS, with clinical microbiological tests will improve the rate of positivity in the diagnosis of rare fungal infections, and the importance of follow-up should be emphasized.

Butenyl-spinosyn is a highly effective and broad-spectrum biopesticide produced by

Reduction and optimization of the microbial genome is an important strategy for constructing synthetic biological chassis cells and overcoming obstacles in natural product discovery and production. However, it is of great challenge to discover target genes that can be deleted and optimized due to the complicated genome of actinomycetes.

To generate a AfsR-like (AfsR-L) overexpression strain Saccharopolyspora pogona-AfsR-L and investigate its effects on the morphology and metabolism of S. pogona. Firstly, we generated the overexpression vector pOJ260-PermE-afsR-L via overlap extension PCR. Then, the recombination strain S. pogona-AfsR-L was constructed via conjugal transfer. To monitor the growth and morphology, mycelia and sporulation were observed. The distinctive proteins and butenyl-spinosyn biosynthesis were investigated by SDS-PAGE, HPLC, and mass spectrometry. And the transcriptional level of afsR-L and other relative functional genes in S. pogona-AfsR-L was analyzed by qRT-PCR. Western blot verified the increased amount of AfsR-L protein in the overexpression strain. Growth curve and mycelia observation showed that afsR-L overexpression make the stationary phase of S. pogona-AfsR-L longer than that of wild S. pogona by approximate 3 days. Moreover, S. pogona-AfsR-L exhibited a more obvious white phenotype on the solid medium, which means afsR-L overexpression affects the sporulation ability of S. pogona. HPLC analysis revealed that the peak area of the butenyl-spinosyn yield of S. pogona-AfsR-L was 293.6, while that of S. pogona was 250.9. SDS-PAGE analysis showed that the two strains had different whole protein expression profiles, and the distinctive proteins were further identified by LC-MS/MS identification, which showed the possible control mechanism of afsR-L gene in S. pogona. We concluded that AfsR could directly or indirectly positively regulate the biosynthesis of butenyl-spinosyn and affect the growth features of S. pogona. We envisioned that this result can be expanded to other Streptomyces for strain improvement.

Background Butenyl-spinosyn, produced by Saccharopolyspora pogona, is a promising biopesticide due to excellent insecticidal activity and broad pesticidal spectrum. Bacterioferritin (Bfr, encoded by bfr) regulates the storage and utilization of iron, which is essential for the growth and metabolism of microorganisms. However, the effect of Bfr on the growth and butenyl-spinosyn biosynthesis in S. pogona has not been explored. Results Here, we found that the storage of intracellular iron influenced butenyl-spinosyn biosynthesis and the stress resistance of S. pogona, which was regulated by Bfr. The overexpression of bfr increased the production of butenyl-spinosyn by 3.14-fold and enhanced the tolerance of S. pogona to iron toxicity and oxidative damage, while the knockout of bfr had the opposite effects. Based on the quantitative proteomics analysis and experimental verification, the inner mechanism of these phenomena was explored. Overexpression of bfr enhanced the iron storage capacity of the strain, which activated polyketide synthase genes and enhanced the supply of acyl-CoA precursors to improve butenyl-spinosyn biosynthesis. In addition, it induced the oxidative stress response to improve the stress resistance of S. pogona. Conclusion Our work reveals the role of Bfr in increasing the yield of butenyl-spinosyn and enhancing the stress resistance of S. pogona, and provides insights into its enhancement on secondary metabolism, which provides a reference for optimizing the production of secondary metabolites in actinomycetes.

Spinosyns, the secondary metabolites produced by

Butenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and broad pesticidal spectrum. However, its synthetic level was low in the wild-type strain. At present, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficiently editing its genome to improve the butenyl-spinosyn yield. To accelerate the genetic modification of S. pogona, we conducted comparative proteomics analysis to screen differentially expressed proteins related to butenyl-spinosyn biosynthesis. A TetR family regulatory protein was selected from the 289 differentially expressed proteins, and its encoding gene (SP_1288) was successfully deleted by CRISPR/Cas9 system. We further deleted a 32-kb polyketide synthase gene cluster (cluster 28) to reduce the competition for precursors. Phenotypic analysis revealed that the deletion of the SP_1288 and cluster 28 resulted in a 3.10-fold increase and a 35.4% decrease in the butenyl-spinosyn levels compared with the wild-type strain, respectively. The deletion of cluster 28 affected the cell growth, glucose consumption, mycelium morphology, and sporulation by controlling the expression of ptsH, ptsI, amfC, and other genes related to sporulation, whereas SP_1288 did not. These findings confirmed not only that the CRISPR/Cas9 system can be applied to the S. pogona genome editing but also that SP_1288 and cluster 28 are closely related to the butenyl-spinosyn biosynthesis and growth development of S. pogona. The strategy reported here will be useful to reveal the regulatory mechanism of butenyl-spinosyn and improve antibiotic production in other actinomycetes. KEY POINTS: • SP_1288 deletion can significantly promote the butenyl-spinosyn biosynthesis. • Cluster 28 deletion showed pleiotropic effects on S. pogona. • SP_1288 and cluster 28 were deleted by CRISPR/Cas9 system in S. pogona.

Summary Butenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and a broad pesticidal spectrum. Currently, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficient understanding of its regulatory mechanism and improving its production by metabolic engineering. Here, we present data supporting a role of the SenX3-RegX3 system in regulating the butenyl-spinosyn biosynthesis. EMSAs and qRT-PCR demonstrated that RegX3 positively controls butenyl-spinosyn production in an indirect way. Integrated proteomic and metabolomic analysis, regX3 deletion not only strengthens the basal metabolic ability of S. pogona in the mid-growth phase but also promotes the flow of the acetyl-CoA produced via key metabolic pathways into the TCA cycle rather than the butenyl-spinosyn biosynthetic pathway, which ultimately leads to continued growth but reduced butenyl-spinosyn production. The strategy demonstrated here may be valuable for revealing the regulatory role of the SenX3-RegX3 system in the biosynthesis of other natural products., Graphical Abstract, Highlights • Butenyl-spinosyn biosynthesis is highly sensitive to Pi control • RegX3 regulates polyP accumulation in S. pogona • RegX3 may indirectly regulate butenyl-spinosyn production • RegX3 plays an important role in the normal growth development of S. pogona, Genetics; Microbiology

Background Saccharopolyspora pogona is a prominent industrial strain due to its production of butenyl-spinosyn, a high-quality insecticide against a broad spectrum of insect pests. TetR family proteins are diverse in a tremendous number of microorganisms and some are been researched to have a key role in metabolic regulation. However, specific functions of TetR family proteins in S. pogona are yet to characterize. Results In the present study, the overexpression of the tetR-like gene sp1418 in S. pogona resulted in marked effects on vegetative growth, sporulation, butenyl-spinosyn biosynthesis, and oxidative stress. By using qRT-PCR analysis, mass spectrometry, enzyme activity detection, and sp1418 knockout verification, we showed that most of these effects could be attributed to the overexpression of Sp1418, which modulated enzymes related to the primary metabolism, oxidative stress and secondary metabolism, and thereby resulted in distinct growth characteristics and an unbalanced supply of precursor monomers for butenyl-spinosyn biosynthesis. Conclusion This study revealed the function of Sp1418 and enhanced the understanding of the metabolic network in S. pogona, and provided insights into the improvement of secondary metabolite production.

Additional file 2: Table S1. Heterologously expressed protein Sp1418 identified by 1D-LC–MS/MS.

Additional file 3: Table S2. Biological insecticidal activity of S. pogona, S. pogona-Δsp1418 and S. pogona-Sp1418.

Additional file 4: Table S3. Primers, plasmids and strains used in this study. Table S4. qRT-PCR primers used in this study.

Additional file 1: Figure S1. Mass spectrum identification of butenyl-spinosyns. MS identification results showed that MS parent ion [M+H]+ = 633 (black arrow) contained 617 (M+H+) (m/z) ion data and a rhamnose ion fragment of 189 molecular mass, which was confirmed as a butenyl-spinosyn component. Figure S2. The sporulation phenotypes in wild type, S. pogona-Δsp1418 and S. pogona-Sp1418. S. pogona-Sp1418 did not produce spores or was almost invisible, and the amount of spores of S. pogona-Δsp1418 is much more than that of S. pogona, but there was no significant difference in the spore morphology of S. pogona and S. pogona-Δsp1418. Figure S3. Tricine-SDS-PAGE analysis of heterologously expressed protein Sp1418. Coomassie Brilliant Blue staining of Tricine-SDS-PAGE showing heterologous protein Sp1418 expressed in the supernatants of E. coli BL21 bearing recombinant plasmid after IPTG induction and ultrasonication. M: 66 kDa protein marker; 1: Samples from E. coli BL21 as a negative control 2: Samples from E. coli BL21 contained the recombinant plasmid. Figure S4. SDS-PAGE gel analysis of total proteins. M: Protein marker; 1: Samples from 96 h S. pogona; 2: Samples from 96 h S. pogona-Sp1418 cells; 3: Samples from 96 h S. pogona-Δsp1418 cells. Compared with the three strains, there are many distinctive bands, and totally 9 proteins were identified via 1D-LC–MS/MS. Figure S5. Construction of pKCcas9d-sgRNA-UHA-DHA and pOJ260-PermE-sp1418. A. Construction of plasmid pKCcas9d-sgRNA-UHA-DHA; B. Construction of plasmid pOJ260-PermE-sp1418.Figure S6. Recombination schematic diagram of vetor pOJ260-PermE-sp1418 and pKCcas9d-sgRNA-UHA-DHA. A. Recombination schematic diagram of vetor pOJ260-PermE-sp1418; B. Recombination schematic diagram of vetor pKCcas9d-sgRNA-UHA-DHA. Figure S7. Identification of S. pogona-Δsp1418 and S. pogona-Sp1418. A: Identification of PermE-sp1418 fragment in S. pogona and S. pogona-Sp1418. M: DL 2000 DNA marker; 1: PCR products of S. pogona with primer pair perm-F/tetR-R; 2: PCR products of S. pogona-Sp1418 with primer pair perm-F/tetR-R; B: PCR amplification of aac(3)IV gene in S. pogona and S. pogona-Sp1418. M: DL 2000 DNA marker; 1–2: PCR products of S. pogona with primer pair Apr-F/Apr-R; 3: PCR products of S. pogona-Sp1418 with primer pair Apr-F/Apr-R; C: Identification of aac(3)IV gene in S. pogona and S. pogona-Δsp1418. M: DL 2000 DNA marker; 1: PCR products of S. pogona with primers Apr-F/Apr-R; 2: PCR products of S. pogona-Δsp1418 with primers Apr-F/Apr-R; D: Identification of sp1418 gene in S. pogona and S. pogona-Δsp1418 M: DL 2000 DNA marker; 1: PCR products of S. pogona with primers tetR-P-F/tetR-P-R; 2: PCR products of S. pogona-Δsp1418 with primers tetR-P-F/tetR-P-R.

Polynucleotide phosphorylase is a highly conserved protein found in bacteria and fungi that can regulate the transcription of related enzymes involved in amino acid metabolism, organic acid metabolism, and cell biosynthesis. We studied the effect of polynucleotide phosphorylase on Saccharopolyspora pogona (S. pogona) growth and the synthesis of secondary metabolites. First, we generated the overexpression vector pOJ260-PermE-pnp via overlap extension PCR. The vector pOJ260-PermE-pnp was then introduced into S. pogona by conjugal transfer, thereby generating the recombination strain S. pogona-Pnp. Results showed that engineering strains possessed higher biomass than those of the wild-type strains. Moreover, the ability of these strains to produce spores on solid medium was stronger than that of the wild-type strains. HPLC results revealed that the butenyl-spinosyn yield in S. pogona-Pnp increased by 1.92-fold compared with that of S. pogona alone. These findings revealed that overexpression of polynucleotide phosphorylase effectively promoted butenyl-spinosyn biosynthesis in S. pogona. This result may be extended to other Streptomyces for strain improvement.

Tumor growth and metastasis depend on angiogenesis. Thus, inhibiting tumor angiogenesis has become promising cancer therapeutic strategy in recent years. Tumstatin is a more powerful angiogenesis inhibitor than endostatin. Anti-angiogenic active fragment encoding amino acids 45-132 (Tum-5) of tumstatin was subcloned into four different inducible expression vectors and successfully solubly expressed in Escherichia coli BL21 (DE3) in this study. Subsequently, an anaerobic inducible expression vector was constructed under Vitreoscilla hemoglobin gene promoter Pvhb in E. coli Nissle 1917 (EcN). The secretory expression of Tum-5 in the engineered bacterium was determined in vitro and in vivo by Western blot or immunochemistry. The anti-tumor effect detection demonstrated that EcN could specifically colonize the tumor, and B16 melanoma tumor growth was remarkably restrained by EcN (Tum-5) in mice bearing B16 melanoma tumor. Abundant infiltrating inflammatory cells were observed in tumor areas of the EcN-treated group through hematoxylin and eosin staining, with a relatively reduced expression of endothelial marker platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) by immunofluorescence in tumor sections of EcN (Tum-5)-treated mice. No significant morphological differences were observed in the liver, kidney and spleen between EcN-treated mice and the control group, indicating that EcN was cleared by the immune system and did not cause systemic toxicity in mice. These findings demonstrated that the gene delivery of Tum-5 to solid tumors could be an effective strategy for cancer therapy.

Previous studies have reported that regulatory T cells (Tregs), which are physiologically engaged in the maintenance of immunological self-tolerance, have a critical role in the regulation of the antitumor immune response. Targeting Tregs has the potential to augment cancer vaccine approaches. The current study therefore aimed to evaluate the role of cytokine-induced killer (CIK) cell infusion in modulating Tregs in patients with non-small cell lung cancer (NSCLC). A total of 15 patients with advanced NSCLC were treated by an infusion of CIK cells derived from autologous peripheral blood mononuclear cells (PBMCs). By using flow cytometry and liquid chip analysis, subsets of T cells and natural killer (NK) cells in peripheral blood, and plasma cytokine profiles in the treated patients, were analyzed at 2 and 4 weeks after CIK cell infusion. Cytotoxicity of PBMCs (n=15) and NK cells (n=6) isolated from NSCLC patients was evaluated before and after CIK cell therapy. Progression-free survival (PFS) and overall survival (OS) were also assessed. Analysis of the immune cell populations before and after treatment showed a significant increase in NK cells (P

The generation of Chinese characters, evolution of Chinese characters and the general linkage between the elements of the historical field have constructed the giant discourse field of the Chinese character. The philosophical construction of discourse field of Chinese character provides a new solution for Chinese character research. This paper is based on the Chinese history and reality, beyond the traditional theory of knowledge platform to examine the key value of Chinese characters, revealing the critical points of Chinese characters formation, ideology and value orientation. The relationship between Chinese character formation and thinking, ideology, culture mode, and academic form in a certain historical context is the basis of reconstructing and locating the value of Chinese philosophy.

Traditional cancer therapies, such as surgery treatment, radiotherapy, and chemotherapy, often fail to completely eliminate tumor cells in an anaerobic microenvironment of tumor regions. In contrast to these traditional cancer therapies, the use of targeted delivery vectors to deliver anticancer genes or antitumor drugs to hypoxic areas in tumors is the most clinically promising cancer treatment with rapid development in recent years. In this study, E.coli Nissle 1917 (EcN), an intestinal probiotic, was utilized as a targeted transport vector to deliver p53 and Tum-5 protein to tumor hypoxic regions. The tumor-targeting characteristics of EcN were investigated using luciferase LuxCDABE operon, and the results demonstrated that EcN could specifically accumulate in the solid tumor areas of SMMC-7721 tumor-bearing BALB/c nude mice. The Tum 5-p53 bifunctional proteins were initially constructed and then delivered to solid tumor regions by using the targeted transporter EcN for cancer therapy. The antitumor effect and safety of three engineered bacteria, namely, EcN (Tum-5), EcN (p53), and EcN (Tum 5-p53), were also examined. The calculated tumor volume and tumor weight indicated that these three engineered bacteria could inhibit the growth of human hepatoma SMMC-7721 cells, and the antitumor effect of EcN (Tum 5-p53) expressing the Tum 5-p53 fusion protein was significantly better than those of EcN (Tum-5) and EcN (p53) alone. Immunofluorescence demonstrated that the expression of Ki-67, a nuclear proliferation-related protein, was inhibited in the tumor areas of the groups treated with the engineered bacteria, whereas the expression of caspase-3 was upregulated. The expression trends of Ki-67 and caspase-3 were consistent with the different antitumor efficacies of these three engineered bacteria. EcN did not elicit obvious side effects on mice. This research not only provids a foundation for tumor-targeted therapy but also contributes greatly to the development of antitumor agents and anticancer proteins. Electronic supplementary material The online version of this article (10.1186/s13036-019-0189-9) contains supplementary material, which is available to authorized users.

This paper presents a longitudinal analysis of the relationship between housing prices and inflation by employing new housing price indices from 29 large Chinese cities over the 2003–2013 period. Based on the Autoregressive Distributive Lag (ARDL) model and bounds test, we find no long-run co-integration relationship between housing prices and inflation. This result is robust for different types of inflation (actual, expected, unexpected inflation). Furthermore, it is found that the housing prices in China grow spectacularly in the sample period owing to the dramatic development of the Chinese economy, while inflation grows in a more modest way. Although the study is conducted in the context of China, the results can provide useful evidence to the debate on the relationship between housing prices and inflation.

Table S1. Primers used in PCR amplification. Figure S1. Agarose gel electrophoresis of human p53 gene amplification products. Figure S2. Construction and identification of recombinant plasmid pET28a-p18. Figure S3. Construction and identification of plasmid pET28a-p18-p53. Figure S4. Purification and identification of recombinant p53 protein. Figure S5. Inhibitory effect of recombinant p53 protein on different tumor cells. Figure S6. The construction program of hypoxia expression vector pET28a-Pvhb-pelB-p18-p53. Figure S7. Construction of p53 hypoxia expression vector pET28a-Pvhb-pelB-p18-p53. Figure S8. Construction of p53 and Tum-5 fusion expression vector. Figure S9. Construction of Tum-5 and p53 fusion expression vector. Figure S10. Morphological changes of different tumor cells after recombinant Tum 5-p53 treated (100Ă ). Figure S11. The construction process diagram of hypoxia expression vector pET28a-Pvhb-pelB-SUMO-Tum 5-MMP-p53. Figure S12. Construction of recombinant plasmid pET28a-Pvhb-pelB-SUMO-Tum 5-MMP-p53 (DOC 1943 kb)

The LytTR family two-component system widely exists in bacterial cells and plays an important role in metabolic regulation. The lytS-L gene that encodes for a LytTR family sensor kinase was knocked out to study its influence on the growth, phenotype, and the biosynthesis of the insecticidal polyketide butenyl-spinosyn in Saccharopolyspora pogona NRRL 30141 (S. pogona). High performance liquid chromatography (HPLC) results showed that the butenyl-spinosyn yield of the lytS-L knockout mutant decreased by 58.9% compared with that of the parental strain. This is manifested by a weak toxicity of the mutant against the insect Helicoverpa assulta (H. armigera). Comparative proteomic analysis revealed the expression characteristics of the proteins in S. pogona and S. pogona-ΔlytS-L: a total of 14 proteins involved in energy metabolism were down-regulated, 9 proteins related to carbon metabolism such as glycolysis, and tricarboxylic acid cycle (TCA) were up-regulated, while 13 proteins involved in the biosynthesis of butenyl-spinosyn were down-regulated (fold change >1.2 or< 0.83). The qRT-PCR (Quantitative Real-time PCR) analysis illustrated that the changes in the expression levels of transcription and translation of the identified genes were consistent. This study explores the function of the two-component system of the LytTR family in S. pogona and shows that the lytS-L gene has an important influence on regulating primary metabolism and butenyl-spinosyn biosynthesis of S. pogona.

Background Syringolin, synthesized by a mixed non-ribosomal peptide synthetase/polyketide synthetase in Pseudomonas syringae pv. syringae (Pss) B728a, is a novel eukaryotic proteasome inhibitor. Meanwhile, directly modifying large fragments in the PKS/NRPS gene cluster through traditional DNA engineering techniques is very difficult. In this study, we directly cloned the syl gene cluster from Pss B301D-R via Red/ET recombineering to effectively express syringolin in heterologous hosts. Results A 22 kb genomic fragment containing the sylA–sylE gene cluster was cloned into the pASK vector, and the obtained recombinant plasmid was transferred into Streptomyces coelicolor and Streptomyces lividans for the heterologous expression of syringolin. Transcriptional levels of recombinant syl gene in S. coelicolor M145 and S. lividans TK24 were evaluated via RT-PCR and the production of syringolin compounds was detected via LC–MS analysis. The extracts of the engineered bacteria showed cytotoxic activity to B16, 4T1, Meth-A, and HeLa tumor cells. It is noteworthy that the syringolin displayed anticancer activity against C57BL/6 mice with B16 murine melanoma tumor cells. Together, our results herein demonstrate the potential of syrinolin as effective antitumor agent that can treat various cancers without apparent adverse effects. Conclusions This present study is the first to report the heterologous expression of the entire syl gene cluster in Streptomyces strains and the successful expression of syringolin in both S. coelicolor M145 and S. lividans TK24. Syringolin derivatives demonstrated high cytotoxicity in vitro and in vivo. Hence, this paper provided an important foundation for the discovery and production of new antitumor compounds.

De novo synthesis of the nucleotide CTP is catalyzed by the essential pyrimidine biosynthesis enzyme CTP synthase (CTPs), which forms large-scale filamentous structures consisting of CTPs termed cytoophidia in prokaryotes and in eukaryotes. Recent studies have shown that cytoophidia are abundant in neuroepithelial stem cells in Drosophila optic lobes and that overexpression of CTPs impairs optic lobe development. Whether CTPs and cytoophidia also play a role in the development of the mammalian cortex remains elusive. Here, we show that overexpression of CTPs by in utero electroporation in the embryonic mouse brain induces formation of cytoophidia in developing cortical neurons and impairs neuronal migration. In addition, the increase of cytoophidia accelerates neuronal differentiation and inhibits neural progenitor cell proliferation by reducing their mitotic activity. Furthermore, we discovered that the cytoophidia diffused during the early G1-phase of the cell cycle. Together, our findings show, for the first time, that CTPs play a significant role in the development of the mammalian cortex.